Biosynthesis of secondary metabolites in the rice blast fungus Magnaporthe grisea: the role of hybrid PKS-NRPS in pathogenicity

mycological research 112 (2008) 207–215

journal homepage: www.elsevier.com/locate/mycres

Biosynthesis of secondary metabolites in the rice blast fungus Magnaporthe grisea: the role of hybrid PKS-NRPS in pathogenicity Je´roˆme COLLEMARE, Alexis BILLARD, Heidi U. BO¨HNERT, Marc-Henri LEBRUN* Centre National de la Recherche Scientifique/UCB/INSA/Bayer CropScience, 14–20 rue Pierre Baizet, 69263 Lyon cedex 09, France

article info

abstract

Article history:

Fungal secondary metabolites are an important source of bioactive compounds for

Received 16 April 2007

agrochemistry and pharmacology. Over the past decade, many studies have been under-

Accepted 9 August 2007

taken to characterize the biosynthetic pathways of fungal secondary metabolites. This ef-

Corresponding Editor:

fort has led to the discovery of new compounds, gene clusters, and key enzymes, and has

Marc Stadler

been greatly supported by the recent releases of fungal genome sequences. In this review, we present results from a search for genes involved in secondary metabolism and their

Keywords:

clusters in the genome of the rice pathogen, Magnaporthe grisea, as well as in other fungal

Biosynthesis

genomes. We have also performed a phylogenetic analysis of recently discovered genes en-

Genomics

coding hybrids between a polyketide synthase and a single non-ribosomal peptide synthe-

Magnaporthe oryzae

tase module (PKS–NRPS), as M. grisea seems rich in these enzymes compared with other

Plant pathology

fungi. Using results from expression and functional studies, we discuss the role of these PKS-NRPS in the avirulence and pathogenicity of M. grisea. ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction Fungal secondary metabolites are often highly bioactive. They include food contaminants that are toxic to animals such as the mycotoxins aflatoxins and trichothecenes produced by Aspergillus spp. and Fusarium spp., respectively (Bennett & Klich 2003), or enzymatic inhibitors such as lovastatin produced by A. terreus (Kennedy et al. 1999). However, their role in fungal biology is still poorly understood. One of the most studied fungal secondary metabolites is the dihydroxy-naphthtalene (DHN) polyketide that is the starting unit of DHN-melanin. This compound is polymerized as a dark brown pigment impregnating fungal cell walls, which protects spores and mycelium against desiccation, oxygen and UV (Henson et al. 1999; Langfelder et al. 2003). Melanin is also required for the turgor build-up in the appressoria of Magnaporthe grisea (Howard & Ferrari 1989; Chumley & Valent 1990) and Colletotrichum lagenarium (Perpetua et al. 1996). This

appressorial turgor allows the fungus to penetrate into host plant tissues (Howard et al. 1991). Melanin-deficient appressoria from these fungal species are unable to retain the osmolytes generating this turgor and therefore cannot penetrate into host tissues. Many secondary metabolites act as pathogenicity factors, such as the host selective toxins (HST) Ttoxin polyketide (C. heterostrophus race T) or AM-toxin cyclic peptide (A. alternata fsp. mali) (Markham & Hille 2001). Among mycotoxins, only the trichothecene deoxynivalenol (DON) produced by Gibberella zeae is clearly involved in pathogenicity (Proctor et al. 1995; Jansen et al. 2005). Deciphering the role in infection of other secondary metabolites produced by plant pathogenic fungi should reveal novel mechanisms involved in the infection process. Fungal secondary metabolites are classified as polyketides, non-ribosomal peptides, terpenes or alkaloids, and are synthesized, respectively, by polyketide synthases (PKS), non-ribsosomal peptide synthetases (NRPS), terpene cyclases

* Corresponding author. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2007.08.003

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(TS), and dimethylallyl diphosphate tryptophan synthases (DMATS). The substrates of PKS and NRPS are, respectively, acyl coenzyme A molecules (mainly malonyl CoA and acetyl CoA) and amino acids (Keller et al. 2005). The substrates of TS are dimethylallyl diphosphate (or isoprene) units, and alkaloids are made from tryptophan and isoprene units (Keller et al. 2005). In fungi, PKS are mainly type I enzymes with several enzymatic domains used iteratively to elongate the polyketide backbone (Fig 1). Fungal NRPS are multifunctional enzymes composed of enzymatic modules used non-iteratively to elongate the amino acid chain (Fig 1).

(Bergmann et al. 2007) and the PKS–NRPS PsoA from A. fumigatus was shown to be involved in the biosynthesis of pseurotin (Fig 3, molecule 9) (Maiya et al. 2007). With the exception of lovastatin (2) and compactin, the metabolites produced by PKS–NRPS are tetramic acids (Royles 1995). Indeed, the NRPS element of the hybrid enzyme is involved in the formation of a peptide bond between a single amino acid and the polyketide synthesized by the PKS module. The intramolecular cyclization of this precursor leads to a tetramic acid (Song et al. 2004). Fungal tetramic acids such as pramanicin (Fig 3, molecule 4) isolated from Stagonospora spp., militarinone (Fig 3, molecule 8) from Paecilomyces militaris, zopfiellamide (Fig 3, molecule 3) from Zopfiella latipes (Song et al. 2004), as well as tenuazonic acid (Fig 3, molecule 10) (Lebrun et al. 1990) and pyrichalasin H (Tsurushima et al. 2005) produced by Magnaporthe grisea, are likely synthesized by PKS–NRPS. The S. nodorum genome contains two PKS–NRPS encoding genes (Fig 2). As only one encodes a full PKS–NRPS (SNOG_00308), the corresponding enzyme is likely involved in the biosynthesis of pramanicin. PKS–NRPS required for the biosynthesis of the other known fungal tetramic acids need to be identified.

PKS–NRPS hybrids: new enzymes involved in secondary metabolite biosynthesis PKS–NRPS hybrids were only recently discovered in fungi (Kroken et al. 2003; Bo¨hnert et al. 2004; Song et al. 2004). These hybrid enzymes consist of a fungal iterative type I PKS fused to a single NRPS module that is sometimes truncated. The phylogenetic analysis of the PKS domains KS and AT shows that they cluster as a single group distinct from other fungal PKS (Kroken et al. 2003; Bo¨hnert et al. 2004) (Fig 2), suggesting a common ancestral ascomycete gene resulting from the fusion between a PKS and a NRPS. An increasing number of fungal PKS–NRPS have been studied for their involvement in the production of secondary metabolites (Fig 3). The LNKS from A. terreus has a truncated NRPS module and is involved in the biosynthesis of the cholesterol-lowering drug lovastatin (Fig 3, molecule 2) (Kennedy et al. 1999). MlcA from Penicillium citrinum, an orthologue of LNKS, is involved in the biosynthesis of compactin, an analogue of lovastatin (Abe et al. 2002). Biosynthesis of the antiviral compound equisetin (Fig 3, molecule 1) requires eqiS from Fusarium heterosporum (Sims et al. 2005). FusS from Gibberella fujikoroi (Song et al. 2004) and TenS from the entomopathogenic fungus Beauveria bassiana (Eley et al. 2007) are involved in the biosynthesis of fusarin C (Fig 3, molecule 5) and tenellin (Fig 3, molecule 6), respectively. More recently, ApdA was found to be responsible for the biosynthesis of aspyridone A (Fig 3, molecule 7) in A. nidulans

Magnaporthe grisea, a model for the study of fungal secondary metabolites and their role in plant–fungal interactions Magnaporthe grisea is an ascomycete fungus responsible for the rice blast disease, causing 10–40 % yield losses worldwide depending on environmental conditions and agronomical practices (Khush 1989). Improving the control of rice blast is of particular importance to help increase rice production in the forthcoming 20 y (Khush 2005). The development of novel control methods using sustainable fungicides and resistant rice cultivars requires a better knowledge of M. grisea pathogenicity factors and rice resistance mechanisms. This is now possible as genomic and molecular tools are available for both rice and M. grisea (Xu et al. 2007). The analysis of the M. grisea genome has highlighted a significant number of genes potentially involved in pathogenicity, such as genes encoding secreted

A KS

B

A

PCP

Module 1 loading

AT

C

A

Module 2

PCP

DH

C

cMeT

A

ER

MeT PCP Module 3

E

KR

ACP

Cy

A

Module 4

TE

PCP TE Release

Fig 1 – Organization of fungal iterative type I PKS and modular NRPS. (A) Functional domains of type I PKS: domains for a minimal PKS (KS, AT, and ACP) are coloured red, domains involved in backbone modification (DH, CMeT, ER, and KR) are coloured green, the domain for release of the synthesized polyketide (TE) is coloured blue. KS, b-ketoacyl CoA synthase; AT, acyl transferase; ACP, acyl carrier protein; DH, dehydratase; cMeT, C-methylase; ER, enoyl reductase; KR, b-keto reductase; TE, thioesterase. (B) Modular organization of a NRPS involved in the synthesis of a tetrapeptide: ‘A’ domains are responsible for the activation of an amino acid and its loading to the corresponding ‘PCP’ domain, ‘C’ domains catalyse peptide bond formation. Additional domains involved in amino acid modification may be present (MeT, E, Cy). The first module is required for the first amino acid loading. The last domain (TE) is involved in the release of the peptide. C, condensation; A, adenylation; PCP, peptidyl carrier protein; MeT, methylation; E, epimerization; Cy, cyclization; TE, thioesterase.

Role of hybrid PKS-NRPS in pathogenicity

97 FOXG 15296 100 GfPKS1 98

GmPKS1 ACLA 023380 CHGG 05358 CHGG 02374 BfPKS5

84

100 100

eqiS

1

Ani fge1 pg C 8000169 AflavusPKS4 GmPKS9 Syn7

80

LNKS

100

100

2

MlcA

ACLA 055680 Syn6 SNOG 00308 4? ACLA 786600 Syn2 CHGG 05286

82 99

Ace1

99

CHGG 01239 SNOG 07866 ChPKS16 Mycgr48870 91 Pa 0240 100 CIMG 06629 UREG 03815 FG 10464 100 ACLA 098920 100 ACLA 077660 96 Ani e gw1 3.1039 Pa 5 6830 Syn4 CHGG 04068 100 FVEG 10497 100 FOXG 11892 BfPKS7 Pa 1 5210 100 ChPKS17 Mycgr86906 100 Trire59315 Necha70660 100

100 100 100

FusS

5

GzPKS10 ncu08399 CHGG 10092

100 Afu8g00540 9 ACLA 004770 ATEG 00325 Syn3 99 Syn8 TenS

6

Trire58285 FOXG 14587 Ao BAE56814 100 AflavusPKS1 7 100 ANapdA NFIA 001530 UREG 06499 AflavusPKS3 XylariaPKS3 100 SS1G 09237 BfPKS6 BfPKS3 Ani gw1 2 86 100 Ani gw1 4 SS1G 13641 BfPKS4 100 AflavusPKS2 Ao BAE65683 100

91

Ani fge1 pg C 13000034 Ao AO090102000166 ACLA 042410 100 NFIA 100520 100 Afu6g13930 ATEG 09617 86 AN1784 Syn5 LDKS 100 mlcB FAS 95 98

0.2

209

proteins or enzymes involved in the biosynthesis of secondary metabolites (Dean et al. 2005). In parallel, significant efforts have been carried out in several laboratories to study these putative pathogenicity factors, including the development of insertion mutant libraries (Jeon et al. 2007) and improved targeted gene replacement methods (Villalba et al. 2007). The protein sequences of the PKS and NRPS catalytic domains are conserved, facilitating their identification in fungal genomes. Conversely, the protein sequences of TS and DMATS are less conserved and their identification is more difficult. Genes encoding proteins involved in the biosynthesis of a secondary metabolite are frequently clustered at a single locus and display the same expression pattern (Keller & Hohn 1997). Therefore, the first step in the identification of fungal secondary metabolites biosynthetic pathways is the retrieval of key genes encoding PKS, NPRS, hybrids, TS, and DMATS. The next step is the characterization of the nearby genes to identify a putative cluster. Analysis of available fungal genomes reveals that ascomycetes have more secondary metabolism genes than basidiomycetes, archeo-ascomycetes, and chytridiomycetes, whereas hemi-ascomycetes and zygomycetes have none (Table 1). Ascomycete genomes contain on average 16 PKS, ten NRPS, two TS, and two DMATS. PKS– NRPS have been identified only in ascomycetes, with an average of three genes per species (Table 1; Fig 2). Neurospora crassa, as well as Coccidioides spp. and its related species Histoplasma capsulatum, have a lower number of PKS (one to nine genes), NRPS (three to six genes) and PKS-NRPS (none to two genes) than other ascomycetes. Five fungal species have more than 40 genes encoding PKS, NRPS, hybrids, TS, and DMATS in their genome, including M. grisea (45 genes, Table 1). M. grisea also has the largest number of PKS–NRPS (a total of nine; ACE1, SYN2-9). Chaetomium globosum is the species most closely related to M. grisea that also has a large number of PKS–NRPS (six in total). Comparison of the PKS–NRPS encoding genes from C. globosum and M. grisea shows that they share only one orthologous gene (ACE1 and CHGG_01239; Fig 2). This observation is also true between other related species such as Fusarium spp. and Aspergillus spp. The PKS–NRPS phylogenetic tree displays 23 families mostly containing very few orthologues (average two to three; Fig 2). PKS–NRPS genes display a discontinuous distribution suggesting numerous duplications and gene losses, as well as recombination events during ascomycete evolution. As plant pathogenic fungi likely produce a significant number of toxins and inhibitors to manipulate or destroy their hosts, it has been proposed that they could have more genes involved in the biosynthesis of

Fig 2 – Phylogenetic tree of KS and AT domains of fungal PKS–NRPS. KS and AT domains of fungal FAS, PKS, and PKS– NRPS were aligned using Clustal W and edited in Genedoc. The phylogenetic tree was constructed using PhyML v2.4.4 (ML algorithm; Guindon & Gascuel 2003) and plotted using MEGA version 3.1 (Kumar et al., 2004). SYN9 (MGG_11086) was not included in this analysis as its KSDAT sequence is only partially determined. However, SYN9 seems closely related to the Neurospora crassa PKS-NRPS ncu08399 according to BlastP analysis and Baker et al. (2006).

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1 Equisetin (Fusarium heterosporum)

6 Tenellin (Beauveria bassiana)

HO

OH

O

N H

O

2 Lovastatin (Aspergillus terreus)

7 Aspyridone A (Aspergillus nidulans)

3 Zopfiellamide A (Zopfiella latipes)

8 Militarinone C (Paecilomyces militaris)

4 Pramanicin (Stagonospora spp.)

9 Pseurotin (Aspergillus fumigatus)

5 Fusarin C (Fusarium moniliforme)

10 Tenuazonic acid (Magnaporthe grisea)

Fig 3 – Secondary metabolites produced by fungal PKS–NRPS.

secondary metabolites than saprobes. This hypothesis seems valid when comparing M. grisea to the saprobe N. crassa (Bo¨hnert et al. 2004; Dean et al. 2005). However, it does not hold true when comparing M. grisea to other saprobes like C. globosum or Aspergillus spp. that have as many secondary metabolism genes as plant pathogenic fungi (Table 1). Therefore, the

abundance of genes involved in secondary metabolism in the genome of a particular fungal species does not seem to correlate with its ecological niche. This observation suggests that saprobes may need as many secondary metabolites as plant pathogens to survive in their environments, most likely in order to compete with other organisms.

Role of hybrid PKS-NRPS in pathogenicity

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Table 1 – Secondary metabolism genes in fungal genomes Fungus

PKS

NRPS

PKS–NRPS hybrids

Sesqui-/Di-terpene cyclases

DMATS

Total

Magnaporthe grisea Fusarium graminearum F. oxysporum F. verticillioides Haematonectria haematococca Trichoderma reesei Chaetomium globosum Neurospora crassa Podospora anserina Botrytis cinerea Sclerotinia sclerotiorum Stagonospora nodorum Mycosphaerella graminicola Cochliobolus heterostrophus Coccicioides imitis

22 13 11 12 12 11 20 6 17 17 16 22 11 23 9

8 12 8 10 8 8 11 3 9 8 5 10 6 9 5

10 2 3 4 1 2 6 1 3 5 2 2 2 2 1

1/2 3/0 0/0 1/0 1/0 1/1 0/1 0/1 0/1 1/4 0/0 1/1 0/1 0/0 0/0

3 0 2 1 1 0 1 1 1 1 1 2 0 0 1

45 30 24 28 23 23 39 12 31 36 24 38 20 34 16

Uncinocarpus reesii Histoplasma capsulatum Aspergillus flavus A. oryzae A. terreus A. niger Neosartorya fischeri Aspergillus fumigatus A. clavatus A. nidulans

6 1 7 30 28 15 16 12 13 26

6 6 15 15 18 12 18 14 12 10

2 0 4 3 3 5 2 2 7 2

0/0 0/0 1/1 1/1 4/1 2/2 1/1 1/1 0/0 2/3

3 1 5 4 4 0 5 3 2 2

17 8 33 54 58 36 43 33 34 45

Saccharomyces cerevisiae Shizosaccharomyces pombe Pichia stipitis Ashbya gossypii Candida albicans C. guilliermondii C. lusitaniae C. tropicalis Lodderomyces elongisporus

0 0 0 0 0 0 0 0 0

0 1 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

0 0 0 0 0 0 0 0 0

0 1 0 0 0 0 0 0 0

Cryptococcus neoformans Ustilago maydis Phanerochaete chrysosporium Laccaria bicolor Coprinopsis cinereus Sporobolomyces roseus

0 3 1 5 2 0

0 3 1 0 1 1

0 0 0 0 0 0

0/0 0/0 0/0 0/0 1/0 0/0

0 0 0 0 1 0

0 6 2 5 5 1

Rhizopus oryzae Phycomyces blakesleeanus Batrachochytrium dendrobatidis

0 0 0

0 0 2

0 0 0

0/0 0/0 0/0

0 0 0

0 0 2

Approximate number of secondary metabolism genes based on BlastP searches at the Broad Institute fungal genome initiative (www.broad.mit. edu/annotation/fgi/) and NCBI database (www.ncbi.nlm.nih.gov) using as queries KS and AT domains from PKS–NRPS ACE1 (Bo¨hnert et al., 2004), MGG_00022.5 (NRPS cyclosporine synthetase) from Magnaporthe grisea, tri5 (XP_383713.1) from Gibberella zeae (sesquiterpene), cps/ks (Q9UVY5) from Gibberella fujikoroi (diterpene) and dmaW (Q12594) from Claviceps fusiformis (DMATS). BlastP searches for Aspergillus fumigatus, A. clavatus, and Neosartorya fischeri were carried out at the TIGR fungal database (www.tigr.org), for A. oryzae at the Central Aspergillus Data Repository (www.cadre-genomes.org.uk), for Podospora anserina at Podospora anserina Genome Project (podospora.igmors.u-psud.fr) and that for Nectria haematococca, Trichoderma reseii, Mycosphaerella graminicola, Pichia stipitis, Phanerochaete chrysosporium, Laccaria bicolor, Sporobolomyces roseus, and Phycomyces blakesleeanus at the DOE Joint Genome Institute (www.jgi.doe.gov). PKS, polyketide synthase; NRPS, non-ribosomal peptide synthetase; DMATS, dimethylallyl tryptophan synthase.

Analysis of the clusters of secondary metabolism genes in M. grisea showed that each of the eight NRPS belongs to a gene cluster, as well as 16 of the 22 PKS. Interestingly, two of these 14 PKS clusters contain two PKS encoding genes. We also found two clusters with two PKS–NRPS among the four clusters with PKS–NRPS. Clusters carrying two PKS genes have been already described in Aspergillus spp. (aflatoxins/sterigmatocystins

clusters with one FAS and one PKS) (Yu et al. 1995; Brown et al. 1996), A. terreus (lovastatin cluster with one truncated PKS–NRPS and one PKS) (Kennedy et al. 1999) and G. zeae (zearalenone cluster with two PKS) (Kim et al. 2005). This particular organization may result from rearrangements between two gene clusters carrying single PKS. Surprisingly, six PKS and three PKS–NRPS-encoding genes do not

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belong to gene clusters in M. grisea. One of these PKS is involved in the biosynthesis of melanin, genes from which are scattered in the M. grisea genome, in contrast to Aspergillus spp. in which they form a cluster (Langfelder et al. 2003).

ACE1, a novel Magnaporthe grisea avirulence gene encoding a PKS–NRPS Resistance of plants to specific races of pathogens often follows a gene-for-gene relationship in which a specific major plant resistance gene (R) is involved in the recognition of pathogens carrying the matching avirulence gene (AVR) (Jones & Dangl 2006). Most fungal AVR genes encode small peptides secreted into host tissues during infection that are recognized either directly or indirectly by the products of the corresponding R genes (Kamoun 2007). ACE1 from Magnaporthe grisea differs from other fungal AVR genes in that it is not a secreted protein (Bo¨hnert et al. 2004), although it behaves as a classical AVR gene. Indeed, M. grisea isolates or transformants carrying the functional AVR gene ACE1 are unable to infect rice cultivars carrying the corresponding R gene Pi33, whereas isolates or mutants defective for ACE1 bypass the rice Pi33-mediated resistance and infect such resistant cultivars. ACE1 was isolated by map-based cloning and encodes a PKS–NRPS involved in secondary metabolism (Bo¨hnert et al. 2004). ACE1 is expressed exclusively in appressoria during fungal penetration of host leaves (Fig 4). The protein Ace1 is only detected in the cytoplasm of the appressoria and not in infectious hyphae differentiated inside infected epidermal cells. Mutation of the putative catalytic

Expression relative to ILV5 (2-

Ct)

3 ACE1 SYN2 SYN6 SYN8

2.5

2

1.5

1

0.5

0 0h

8h

17h

24h

Appressorial Penetration differentiation and maturation

30h

48h

52h mycelium

Colonization

Fig 4 – Expression pattern of Magnaporthe grisea PKS-NRPS genes during infection and mycelium growth. The expression of M. grisea PKS-NRPS genes was assessed by real-time RT-PCR using RNA extracted from infected barley leaves at different times during the infection, and from Guy11 mycelium grown in complete medium. Gene expression is relative to ILV5 expression according to the formula 2L(Ct gene X L Ct ILV5) (Livak & Schmittgen 2001).

site of the b-ketoacyl synthase domain of Ace1 abolishes recognition of the fungus by resistant rice, suggesting that Ace1 biosynthetic activity is required for avirulence. Altogether, these results strongly suggest that the avirulence signal recognized by Pi33 rice cultivars is not the Ace1 protein, but the secondary metabolite synthesized by Ace1. According to this hypothesis, rice resistant plants have evolved mechanisms to recognize M. grisea through the perception of one of its secondary metabolites. This phenomenon suggests that plants sense a large array of pathogen-associated signals (Ingle et al. 2006) to protect themselves from microbial attacks.

Are Magnaporthe grisea PKS–NRPS involved in pathogenicity? The genome of Magnaporthe grisea contains eight other PKS– NRPS genes besides ACE1. Why are PKS–NRPS are so abundant in the genome of M. grisea? It is tempting to speculate that this higher number results from an evolutionary trend of this plant pathogenic species towards an increase in its number of secondary metabolites as they may help colonize novel host plants. To address this question, we first assessed the expression of M. grisea PKS–NRPS genes during the infection of barley leaves by the wild-type isolate Guy11. Real-time RT-PCR experiments were carried out using RNA extracted from infected barley leaves at different time points after inoculation and from Guy11 mycelium grown in vitro (Fig 4). SYN9 (MGG_11086.5) (Baker et al. 2006) was not included in this study as this gene is only partially sequenced. The expression of SYN3, SYN4, SYN5 and SYN7 was not detected in the conditions tested (barley leaf infection and in vitro grown mycelium). These four genes may be expressed under particular conditions, such as nutrient starvation and other stresses or at particular developmental stages, as observed in Aspergillus spp. with sporulation (Calvo et al. 2002). The four other PKS–NRPS genes are expressed during barley leaf infection (Fig 4). ACE1 and SYN2 expression is detected as early as 8 h after infection (hai), whereas SYN8 expression is first detected at 17 hai. The expression of these three genes reaches a maximum at 17 hai, and then subsequently decreases rapidly, and the genes are not expressed at 48 hai. SYN2 is five times less expressed than ACE1, and SYN8 a hundred times less than ACE1. These three genes are not expressed in the mycelium and are therefore specifically expressed during the penetration of the fungus into host leaves. The fourth PKS–NRPS gene, SYN6, is expressed in mycelium grown in vitro and is expressed during infection (Fig 4). The expression pattern of SYN6 suggests a role in colonization and that of ACE1, SYN2, and SYN8 suggests that secondary metabolism is active in the appressoria during the penetration of host tissues. As penetration of M. grisea into host plants is mostly mechanical and only requires mature melanized appressoria with high turgor, it is unlikely to involve secondary metabolites, beside melanin. However, these metabolites may collectively inhibit the metabolism and defence reactions of the first epidermal cell, facilitating the early establishment of the fungus in its host plant. Deletions of ACE1, SYN2, and SYN6 were performed to assess their role in the infection process. Dace1 deletion mutants are as pathogenic as the corresponding wild-type isolate Guy11 on

Role of hybrid PKS-NRPS in pathogenicity

susceptible rice cultivars that do not carry the Pi33 resistance gene, demonstrating that the metabolite synthesized by Ace1 is not essential for M. grisea infection (Bo¨hnert et al. 2004). Knock-out mutants were also obtained for SYN2 (Collemare et al., unpubl.) and SYN6 (unpubl.) in Guy11 and Guy11Dku80 background (Villalba et al. 2007), respectively. Both Dsyn2 and Dsyn6 mutants are as pathogenic as wild-type isolates on susceptible rice cultivars. The metabolites synthesized by these three PKS–NRPS are therefore not essential for the infection of the fungus into host plants or are functionally redundant.

A complex regulation of PKS–NRPS gene expression in Magnaporthe grisea? ACE1 expression pattern was extensively studied using a transcriptional fusion between eGFP and ACE1 promoter and terminator (Bo¨hnert et al. 2004; Fudal et al. 2007). ACE1 is only expressed in appressoria differentiated on plant leaves or artificial cellophane membranes when the appressorium starts to initiate the penetration process. In contrast, it is only expressed at a low level (1–5 % of the appressoria) in appressoria differentiated on artificial membranes that cannot be pierced by the fungus (Teflon (Goodfellow, Cambridge, UK), Mylar (Rhodia, Lyon, France)). ACE1 expression is not induced by plant cell wall compounds or cellophane membrane extracts and is independent of the cAMP pathway (Mitchell & Dean 1995; Adachi & Hamer 1998) and Pls1 (Clergeot et al. 2001), both of which are required for appressorium-mediated penetration (Fudal et al. 2007). It was shown that ACE1 is not expressed in the appressoria of the melanin-defective mutant, buf1, except when adding hyper-osmotic solutes that restore appressorial development. Treatment of Magnaporthe grisea appressoria differentiated on onion epidermis with actin or tubulin inhibitors abolishes in a dose-dependant manner both fungal penetration and ACE1 expression. These experiments demonstrate that appressorium-specific expression of ACE1 does not depend on host plant signals, but is connected to the onset of appressorium mediated penetration. Targeted gene replacement at the ACE1 locus was not efficient in wild-type isolate Guy11 (1.5 % of transformants) (Bo¨hnert et al. 2004). This rate was not increased in the Guy11-Dku80 strain in which targeted gene replacement usually reaches 80–100 % (Villalba et al. 2007). A similar low homologous recombination rate was observed when attempting to delete SYN2 (Collemare et al. unpubl.) located close to ACE1 (42 kb upstream). These results suggest that the region carrying ACE1 has a reduced ability for homologous recombination. Chromosomal regions carrying clusters of secondary metabolism genes may have a particular chromatin conformation. Indeed in Aspergillus spp., the expression of genes from the aflatoxin cluster requires the histone methyl transferase LaeA (Bok & Keller 2004), which behaves as a global regulator of gene clusters involved in secondary metabolism (Bok et al. 2006). LaeA likely modifies chromatin conformation of loci carrying secondary metabolism gene clusters, increasing their expression by facilitating the access of regulatory proteins to DNA. ACE1 and SYN2 could be controlled by a similar epigenetic

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mechanism involving a particular chromatin conformation when not expressed. This chromatin status at the ACE1 locus may limit the access to DNA of proteins involved in recombination (Bao & Shen 2007), lowering targeted gene replacement frequencies.

Conclusions The secondary metabolite synthesized by the PKS–NRPS Ace1 in Magnaporthe grisea likely acts as an avirulence signal recognized by rice cultivars carrying the Pi33 resistance gene (Bo¨hnert et al. 2004). The Ace1 metabolite is the only example of a fungal secondary metabolite that is recognized by resistant host plants. However, the primary biological role of the metabolite produced by Ace1 is certainly not conferring avirulence. It has been hypothesized that avirulence factors are pathogenicity effectors that have been recognized by plants during their evolution towards resistance against pathogens (Jones & Dangl 2006). As secondary metabolites are known for their biological activities, mostly as inhibitors or toxins such as HSTs (Markham & Hille 2001), metabolites produced by M. grisea PKS–NRPS could behave as toxins killing infected cells or suppressing host defences. Among the nine PKS–NRPS genes identified in the M. grisea genome, four (ACE1, SYN2, SNY6, and SYN8) are specifically expressed or over-expressed during infection. The tight appressorium-specific expression of ACE1, SYN2, and SYN8 suggests that they have a role during the penetration of or settling in first infected cells, whereas the SYN6 expression profile suggests a role during the colonization of host tissues by the fungus. However, single-deletion mutants (ACE1, SYN2, or SYN6) were still pathogenic on rice. This discrepancy may result from a functional redundancy between the metabolites from Ace1, Syn2, and Syn8 that are produced at the same stage of infection and may have a similar toxic effect on host plants. Accordingly, the absence of a single metabolite may not be sufficient to reduce significantly the pathogenicity of the corresponding mutant. Double and triple mutants are needed to assess this hypothesis. If the role of M. grisea PKS–NRPS in infection cannot be assessed by genetic methods, it is important to identify the secondary metabolites produced by these enzymes. Indeed, the characterization of their activities on rice and other plants may suggest a role in infection. If the metabolites produced by Ace1, Syn2, Syn8 and Syn6 have a high toxicity towards plants, it would provide additional clues for their role as toxins in the pathogenicity of M. grisea. Such identification is underway in our laboratory and we have developed strategies based on LC/MS (Liquid Chromatography/Mass Spectrometry) analysis of culture filtrates from PKS–NRPS over-expressing transformants or deletion mutants. Another strategy is the over-expression of the cluster-specific regulator that was successfully used to characterize aspyridone A produced by Aspergillus nidulans (Bergmann et al., 2007). Characterization of novel fungal secondary metabolites is an exciting field from the point of view of plant pathologists, biochemists and chemists. Indeed, the study of secondary metabolism may help to

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discover novel compounds with interesting structures or modes of action that could be useful for agrochemistry or pharmacology.

Acknowledgements We would like to thank M. Viaud (PMDV-INRA, Versailles) and P. Silar (Institut de Ge´ne´tique Mole´culaire, Orsay) for PKS-NRPS sequences of Botrytis cinerea and Sclerotinia sclerotiorum, and Podospora anserina, respectively. We also thank Z. Song, R. Cox and T. Simpson (School of Chemistry, University of Bristol) and C. Lazarus (School of Biological Sciences, University of Bristol) for their collaboration in characterizing the metabolite produced by Ace1, and for helpful discussion.

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